Transthyretin quaternary and tertiary structural changes facilitate misassembly into amyloid.

Human transthyretin (TTR) can be transformed into amyloid fibrils by partial acid denaturation to yield a monomeric amyloidogenic intermediate that self-associates into amyloid through quaternary structural intermediates, which are identified by sedimentation velocity methods. The monomeric amyloidogenic intermediate has substantial beta-sheet structure with a nonnative but intact tertiary structure as discerned from spectroscopic methods. Proteolysis sensitivity studies suggest that the C-strand-loop-D-strand portion of TTR becomes disordered and moves away from the core of the beta-sandwich fold upon formation of the monomeric amyloidogenic intermediate over the pH range 5.1-3.9. The single site mutations that are associated with early onset amyloid disease [familial amyloid polyneuropathy (FAP)] function by destabilizing tetrameric TTR. Under mild denaturing conditions, the FAP variants populate the monomeric amyloidogenic intermediate conformation, which assembles into amyloid, whereas wild-type TTR remains tetrameric and nonamyloidogenic. The FAP mutations do not significantly alter the native folded structure; instead, they appear to act by making the thermodynamics and perhaps the kinetics more favorable for formation of the amyloidogenic intermediate. Suppressor mutations have also been characterized that strongly stabilize tetrameric TTR and disfavor the formation of the monomeric amyloidogenic intermediate, thus inhibiting amyloid formation. The mechanistic details characterizing transthyretin amyloid fibril formation available from the biophysical studies outlined within have been utilized to develop a new therapeutic strategy for intervention in human amyloid disease. This approach features small molecules that bind with high affinity to the normal fold of transthyretin, inhibiting the quaternary and tertiary structural changes associated with the formation of the monomeric amyloidogenic intermediate that self-assembles into amyloid. Ligand binding to TTR stabilizes the native tetrameric fold, which is nonamyloidogenic.

FAP mutations destabilize transthyretin facilitating conformational changes required for amyloid formation.

Functional transthyretin (TTR) can be transformed into amyloid by partial acid denaturation yielding a monomeric amyloidogenic intermediate which self-associates. The amyloidogenic intermediate has substantial beta-sheet structure with non-native but defined tertiary structure. pH-dependent proteolysis sensitivity studies have identified portions of TTR which become disordered and solvent-exposed in the amyloidogenic intermediate. These include the C-strand-loop D-strand portion of TTR which moves away from the core of the beta-sandwich fold. Mutations that are associated with early onset-amyloid disease (familial amyloidotic polyneuropathy; FAP) function by destabilizing tetrameric TTR in favour of the monomeric amyloidogenic intermediate which has a rearranged C-strand-loop D-strand region. In most cases the FAP mutations do not significantly alter the native folded structure, but instead act on the denaturation pathway by a mechanism that is not completely understood. Interestingly, mutations have also been characterized which strongly stabilize tetrameric TTR and make amyloid formation very difficult at pHs accessible in vivo.

Comparison of lethal and nonlethal transthyretin variants and their relationship to amyloid disease.

The role that transthyretin (TTR) mutations play in the amyloid disease familial amyloid polyneuropathy (FAP) has been probed by comparing the biophysical properties of several TTR variants as a function of pH. We have previously demonstrated that the partial acid denaturation of TTR is sufficient to effect amyloid fibril formation by self-assembly of a denaturation intermediate which appears to be monomeric. Earlier studies on the most pathogenic FAP variant known, Leu-55-Pro, revealed that this variant is much less stable toward acid denaturation than wild-type TTR, apparently explaining why this variant can form amyloid fibrils under mildly acidic conditions where wild-type TTR remains nonamyloidogenic. The hypothesis that FAP mutations destabilize the TTR tetramer in favor of a monomeric amyloidogenic intermediate under lysosomal (acidic) conditions is further supported by the data described here. We compare the acid stability and amyloidogenicity of the most prevalent FAP variant, Val-30-Met, along with the double mutant, Val-30-Met/Thr-119-Met, which serves to model the effects of these mutations in heterozygous patients where the mutations are in different subunits. In addition, we have characterized the Thr-119-Met TTR variant, which is a common nonpathogenic variant in the Portuguese population, to further investigate the role that this mutation plays in protecting individuals who also carry the Val-30-Met mutation against the classically severe FAP pathology. This biophysical study demonstrates that Val-30-Met TTR is significantly less stable toward acid denaturation and more amyloidogenic than wild-type TTR, which in turn is less stable and more amyloidogenic than Thr-119-Met TTR. Interestingly, the double mutant Val-30-Met/Thr-119-Met is very similar to wild-type TTR in terms of its stability toward acid denaturation and its amyloidogenicity. The data suggest that the Thr-119-Met mutation confers decreased amyloidogenicity by stabilizing tetrameric TTR toward acid denaturation. In addition, fluorescence studies monitoring the acid-mediated denaturation pathways of several TTR variants reveal that the majority exhibit a plateau in the relative fluorescence intensity over the amyloid-forming pH range, i.e., ca. pH 4.3-3.3. This intensity plateau suggests that the amyloidogenic intermediate(s) is (are) being observed over this pH range. The Thr-119-Met variant does not exhibit this plateau presumably because the amyloidogenic intermediate(s) do(es) not build up in concentration in this variant. The intermediate is undoubtedly forming in the Thr-119-Met variant, as it will form amyloid fibrils at high concentrations; however, the intermediate is only present at a low steady-state concentration which makes it difficult to detect.

Amino acids that specify structure through hydrophobic clustering and histidine-aromatic interactions lead to biologically active peptidomimetics.

Acyclic beta-sheet structure can be nucleated in heptapeptides when the 4-(2-aminoethyl)-6-dibenzofuranpropanoic acid residue (1) is flanked in sequence by two His residues, a His residue and a hydrophobic residue or by two hydrophobic residues. Acyclic beta-sheet peptidomimetics having an appropriate sequence have sufficient structural integrity to exhibit antimicrobial activity equivalent to that of gramicidin S.

Intermolecular disulfide linkages are not required for transthyretin amyloid fibril formation in vitro.

The role of intermolecular disulfide linkages in transthyretin (TTR) amyloid fibril formation was investigated by comparing wild type TTR to Cys-10-Ala TTR which is incapable of disulfide formation. The Cys-10-Ala variant exhibits quaternary structural stability equal to the wild type protein under acidic denaturing conditions. Both Cys-10-Ala and wild type TTR were converted into amyloid fibrils by partial acid denaturation. There was no evidence of intermolecular disulfide formation in the case of wild type amyloid fibrils. These results are inconsistent with a recently proposed model stressing the importance of intermolecular disulfide linkages in TTR amyloid fibril formation, but are consistent with a model relying on noncovalent quaternary contacts made possible through an acid-mediated conformational change.

Transthyretin mutation Leu-55-Pro significantly alters tetramer stability and increases amyloidogenicity.

A recently reported variant of human transthyretin (TTR), Leu-55-Pro, implicated as the causative agent in early-onset familial amyloid polyneuropathy was expressed and characterized, and its denaturation pathway and amyloidogenicity were compared to those of wild-type transthyretin. The overlap-extension polymerase chain reaction (PCR) methodology was used to introduce the Leu-55-Pro mutation into the transthyretin DNA sequence and to construct a new expression system. The Leu-55-Pro variant of transthyretin was expressed with a leader sequence to ensure secretion into the periplasmic space of Escherichia coli. Transthyretin's resistance to sodium dodecyl sulfate- (SDS-) induced denaturation was utilized to measure the quaternary stability as a function of pH employing SDS-polyacrylamide gel electrophoresis (PAGE) in the presence and absence of an amyloid fibril inhibitor, Z 3-14. These studies reveal that the Leu-55-Pro TTR tetramer is significantly less stable than wild-type TTR. This is relevant because we have previously shown that the partial denaturation of transthyretin is sufficient to effect amyloid fibril formation from a denaturation intermediate which may be a structured monomer. The ability of Leu-55-Pro TTR to denature to the amyloidogenic intermediate at pHs where the wild-type protein is stable may explain the variant's propensity to form amyloid fibrils in vitro and in vivo where the wild-type protein remains stable and nonamyloidogenic. Congo red binding, polarized light microscopy, and electron microscopy confirm the characteristic structure of amyloid fibrils produced from Leu-55-Pro TTR in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)